22. 10Â phosphate buffer: Dissolve 60.4 g Na 2 HPO 4 and 9.0 g
Na 2 HPO 4 in DI water. Take volume to 0.5 L. Autoclave using
a liquid cycle for 20 min. Store at room temperature.
23. 0.2% Tween 80 solution. Add 20 mL 10% Tween 80 to
980 mL DI water. Autoclave using a liquid cycle for 20 min.
Store at room temperature.
24. Buffered water (0.1% Tween 80, 0.1 M phosphate). To
800 mL DI water, add 100 mL of 10Â phosphate buffer (see
above) and 10 mL of 10% Tween 80. Adjust pH to 7.1–7.2.
Complete to 1 L with DI water. Autoclave using a liquid cycle
for 20 min. Store at room temperature.
25. 0.01 M phosphate saline buffer: To 800 mL DI water, add
1.09 g KH 2 PO 4 . Adjust pH to 6.9. Dissolve 0.46 g NaCl.
26. 6% PEG/8% Dextran/0.01 M phosphate saline buffer: To
800 mL of 0.01 M phosphate saline buffer, add 48.0 g of
polyethylene glycol (PEG) and 64 g of dextran. Scoop dextran
and add slowly to the PEG/potassium phosphate solution
(do not pour in one go) for complete dissolution. DO NOT
complete the volume. Autoclave using a liquid cycle for
20 min. Store at room temperature. Solution will be clear
after autoclaving; shaking will cause cloudiness and separation
into layers.
27. 1 M calcium chloride. Dissolve 27.75 g of CaCl 2 in DI water,
and then complete to a final volume of 250 mL. Autoclave
using a liquid cycle for 20 min. Store at 4
C.
28. Collagenase (lyophilized powder, from sterile-filtered
solution).
29. Trypsin (lyophilized powder, bioreagent or cell culture grade).
30. Chymotrypsin (lyophilized powder, cell culture or USP grade).
31. 0.2 μm Acrodisc syringe filters.
32. 1 mL syringes.
33. 500 mL separatory funnel.
34. Microslides.
35. Microscope.
36. Glass tubes (see Note 14).
37. Spectrometer.
2.14 Culture
of M. tuberculosis
Using a Fermentor/
Bioreactor Under
Normoxic and Hypoxic
Conditions
1. GAS media (see Subheading 2.8, item 6 and see Note 15).
2. Bioreactor compatible for the use of BioBLU fermentation
vessels (see Note 16).
3. Two BioBLU, single-use bioreactor vessel, single-pitched
blade with up to 3.75 L working volume capacity and macrosparger (from Eppendorf North America, Inc).
30
Elizabeth Wallace et al.
Na 2 HPO 4 in DI water. Take volume to 0.5 L. Autoclave using
a liquid cycle for 20 min. Store at room temperature.
23. 0.2% Tween 80 solution. Add 20 mL 10% Tween 80 to
980 mL DI water. Autoclave using a liquid cycle for 20 min.
Store at room temperature.
24. Buffered water (0.1% Tween 80, 0.1 M phosphate). To
800 mL DI water, add 100 mL of 10Â phosphate buffer (see
above) and 10 mL of 10% Tween 80. Adjust pH to 7.1–7.2.
Complete to 1 L with DI water. Autoclave using a liquid cycle
for 20 min. Store at room temperature.
25. 0.01 M phosphate saline buffer: To 800 mL DI water, add
1.09 g KH 2 PO 4 . Adjust pH to 6.9. Dissolve 0.46 g NaCl.
26. 6% PEG/8% Dextran/0.01 M phosphate saline buffer: To
800 mL of 0.01 M phosphate saline buffer, add 48.0 g of
polyethylene glycol (PEG) and 64 g of dextran. Scoop dextran
and add slowly to the PEG/potassium phosphate solution
(do not pour in one go) for complete dissolution. DO NOT
complete the volume. Autoclave using a liquid cycle for
20 min. Store at room temperature. Solution will be clear
after autoclaving; shaking will cause cloudiness and separation
into layers.
27. 1 M calcium chloride. Dissolve 27.75 g of CaCl 2 in DI water,
and then complete to a final volume of 250 mL. Autoclave
using a liquid cycle for 20 min. Store at 4
C.
28. Collagenase (lyophilized powder, from sterile-filtered
solution).
29. Trypsin (lyophilized powder, bioreagent or cell culture grade).
30. Chymotrypsin (lyophilized powder, cell culture or USP grade).
31. 0.2 μm Acrodisc syringe filters.
32. 1 mL syringes.
33. 500 mL separatory funnel.
34. Microslides.
35. Microscope.
36. Glass tubes (see Note 14).
37. Spectrometer.
2.14 Culture
of M. tuberculosis
Using a Fermentor/
Bioreactor Under
Normoxic and Hypoxic
Conditions
1. GAS media (see Subheading 2.8, item 6 and see Note 15).
2. Bioreactor compatible for the use of BioBLU fermentation
vessels (see Note 16).
3. Two BioBLU, single-use bioreactor vessel, single-pitched
blade with up to 3.75 L working volume capacity and macrosparger (from Eppendorf North America, Inc).
30
Elizabeth Wallace et al.